Objective
To study the effects of several neurotrophic factors and growth factors on the survival of human retinal ganglion cells(RGC)in vitro.
Methods
RGC were isolated from donor eyes and cultured.RGC in cell culture were identified by morphologic criteria and immunocytochemical staining.Various neurotrophic factors and growth factors were added individually to the cultures.Numbers of RGC in wells in which these agents had been added were compared with those from control wells(cultures without supplements).
Results
No or very few RGC were present in cell cultures containing medium without supplements or those supplemented with neurotrophin-3(NT-3),nerve growth factor (NGF),epidermal growth factor(EGF)amd plateletderived growth factor(PDGF).Numbers of RGC(per 10 fields)in cell cultures containing brain derived neurotrophic factor(BDNF),ciliary neurotrophic factor(CNTF),neurotrophin-4/5(NT-4/5)and basic fibroblast growth factor(bFGF)wer 4.08,1.23,2.63 and 2.65,respectively,significantly more than found in the control cultures.
Conclusions
BDNF,NT-4/5,bFGF,CNTF improve survival of human RGC in vitro,while NGF,NT-3,EGF and PDGF do not.
(Chin J Ocul Fundus Dis, 1999, 15: 149-152)
ObjectiveTo observe the effect of transplantation of neural stem cells (NSCs) induced by all-trans-retinoic acid (ATRA) combined with glial cell line derived neurotrophic factor (GDNF) and chondroitinase ABC (ChABC) on the neurological functional recovery of injured spinal cord in Sprague Dawley (SD) rats.
MethodsSixty adult SD female rats, weighing 200-250 g, were randomly divided into 5 groups (n=12): sham operation group (group A), SCI model group (group B), NSCs+GDNF treatment group (group C), NSCs+ChABC treatment group (group D), and NSCs+GDNF+ChABC treatment group (group E). T10 segmental transversal injury model of the spinal cord was established except group A. NSCs induced by ATRA and marked with BrdU were injected into the site of injury at 8 days after operation in groups C-E. Groups C-E were treated with GDNF, ChABC, and GDNF+ChABC respectively at 8-14 days after operation;and group A and B were treated with the same amount of saline solution. Basso Beattie Bresnahan (BBB) score and somatosensory evoked potentials (SEP) test were used to study the functional improvement at 1 day before remodeling, 7 days after remodeling, and at 1, 2, 5, and 8 weeks after transplantation. Immunofluorescence staining and HE staining were performed to observe the cells survival and differentiation in the spinal cord.
ResultsFive mouse died but another rats were added. At each time point after modeling, BBB score of groups B, C, D, and E was significantly lower than that of group A, and SEP latent period was significantly longer than that of group A (P<0.05), but no difference was found among groups B, C, D, and E at 7 days after remodeling and 1 week after transplantation (P>0.05). BBB score of groups C, D, and E was significantly higher than that of group B, and SEP latent period was significantly shorter than that of group B at 2, 5, and 8 weeks after transplantation (P<0.05);group E had higher BBB score and shorter SEP latent period than groups C and D at 5 and 8 weeks, showing significant difference (P<0.05). HE staining showed that there was a clear boundary between gray and white matter of spinal cord and regular arrangement of cells in group A;there were incomplete vascular morphology, irregular arrangement of cells, scar, and cysts in group B;there were obvious cell hyperplasia and smaller cysts in groups C, D, and E. BrdU positive cells were not observed in groups A and B, but could be found in groups C, D and E. Group E had more positive cells than groups C and D, and difference was significant (P<0.05). The number of glial fibrillary acidic protein positive cells of groups C, D, and E was significantly less than that of groups A and B, and it was significantly less in group E than groups C and D (P<0.05). The number of microtubule-associated protein 2 positive cells of groups C, D, and E was significantly more than that of groups A and B, and it was significantly more in group E than groups C and D (P<0.05).
ConclusionThe NSCs transplantation combined with GDNF and ChABC could significantly promote the functional recovery of spinal cord injury, suggesting that GDNF and ChABC have a synergistic effect in the treatment of spinal cord injury.
摘要:目的:觀察阿托伐他丁對腦梗死大鼠腦保護的作用以及對腦源性神經營養因子(braindeprived neurotrophic factor,BDNF)的影響。方法: 線栓法制備SD大鼠大腦中動脈梗死(middle cerebral artery occlusion,MCAO)再灌注模型。將大鼠隨機分為:假手術組;MCAO組的2 h、24 h、3 d、5 d組;阿托伐他丁組的2 h、24 h、3 d、5 d組。MCAO組和阿托伐他丁組的各時程組再分別分為腦梗死體積亞組、免疫組化亞組,每亞組及假手術組各6只大鼠。在不同時間點觀察阿托伐他丁組和MCAO組大鼠神經行為評分、腦梗死體積,用免疫組化法檢測BDNF陽性細胞數。結果: 神經行為評分和腦梗死體積在阿托伐他丁組和MCAO組的2 h組之間無顯著性差異(Pgt;0.05),在阿托伐他丁24 h、3 d、5 d組均顯著低于對應時程的MCAO組(Plt;0.05);各組缺血半暗帶BDNF陽性細胞數均增高,但阿托伐他丁組的陽性細胞數顯著高于對應時程的MCAO組(Plt;0.05)。結論:阿托伐他丁能提高大鼠局灶腦缺血半暗帶BDNF的表達水平,促進神經元的修復。Abstract: Objective: To observe the effect of atorvastatin in cerebral protection and braindeprived neurotrophic factor(BDNF) in rats. Methods: Ischemic reperfusion model of rats as established by an intraluminal filament and recirculation at different time point respectively. One hundred and two healthy SD rats were randomly assigned into three groups for different preconditioning, including the sham surgery group (SS, n=6), the sham and middle cerebralartery occlusion (MCAO) group (MCAO, n=48), and the atorvastatin and MCAO group (atorvastatin +MCAO, n=48). The latter two groups were further divided into two subgroups on different time points of tests. Each subgroup hase six rats. In the atorvastatin +MCAO group, intragastric administration of atorvastatin was given for five days, then the MCAO followed. In the MCAO group, the MCAO was given directly. The neurophysical marks and the volume of the cerebral infarction in atorvastatin group and MCAO group were determined at different time point. The expression of BDNF was valued by immunohistochemitry respectively. Results: At 2 h, there were no differences in the neurophysical marks and volume of the cerebral infarction between atorvastatin group and MCAO group (Pgt;0.05). At 24 h,3 d,5 d, the neurophysical marks and volume of the cerebral infarction of atorvastatin group were lower than that of MCAO group in the corresponding time (Plt;0.05). Around the necrotic areas,BDNF positive neurons were increased in both groups, but they were higher in atorvastatin group than in MCAO group in the corresponding time (Plt;0.05). Conclusion: Atorvastatin could increase the expression level of BDNF and promote the ischemic neuron to revive.
OBJECTIVE To investigate the effects of targeted muscular injection of ciliary neurotrophic factor (CNTF) on the regeneration of injured peripheral nerves. METHODS The left sciatic nerves of 80 Sprague-Dawley rats were excised to form 6 mm defect and the two ends were bridged by silicone tubes, they were randomly divided into two groups, CNTF group and normal saline (NS) group. The CNTF group was given recombinant human CNTF, 1 mg/kg every other day for 30 days, and the NS group was given equal quantity of normal saline as NS group. The sciatic nerve functional index (SFI), electrophysiological assessment, morphometric analysis of axons, and choleratoxin horseradish peroxidase (CB-HRP) retrograde-labelling were measured postoperatively. RESULTS The SFI, electrophysiological parameters (nerve conduction velocity, latency and amplitude of compound muscle action potentials), myelinated axons counts, mean axons diameters and myelin sheath thickness, number of CB-HRP labelled ventral horn motor neurons of spinal cord were significantly higher in CNTF group than that of NS group. CONCLUSION Targeted muscular injection of CNTF can promote the regeneration of peripheral nerve and improve the nerve functional recovery.
Objective To investigate the memory amelioration of the Alzheimer disease (AD)model rat after being transplanted the single neural stem cells(NSC) and NSC modified with human brain-derived neurotrophic factor（hBDNF） gene. Methods Forty SD rats were divided evenly into 4 groups randomly. The AD model rats were made by cutting unilaterallythe fibria fornix of male rats. Ten to twelve days after surgery, the genetically modified and unmodified NSC were implanted into the lateral cerebral ventricle of group Ⅲ and group Ⅳ respectively. Two weeks after transplantation, theamelioration of memory impairment of the rats was detected by Morris water maze. Results The average escaping latency of the group Ⅲ and group Ⅳ (41.84±21.76 s,25.23±17.06 s respectively) was shorter than that of the group Ⅱ(70.91±23.67 s) (Plt;0.01). The percentage of swimming distance inthe platform quadrant in group Ⅲ (36.9%) and in group Ⅳ(42.0%) was higherthan that in the group Ⅱ(26.0%) (Plt;0.01). More marginal and random strategies were used in group Ⅱ.The percentage of swimming distance in the platform quadrant in group Ⅳ was also greater than that in group Ⅲ(Plt;0.05). There were no significant differences in the average escaping latency, the percentage of swimming distance in the platform quadrant and the probe strategy between group Ⅳ and group Ⅰ(Pgt;0.05).More lineal and oriented strategies were used in group Ⅳ. Conclusion The behavioral amelioration of AD model rat was obtained by transplanting single NSC and hBDNF-gene-modified NSC. The effect of the NSC group modified with hBDNF gene is better than that of the groupⅢ.
Objective To investigate the mRNA expression of ciliary neurotrophic factor on the retina during injury and repair of optic nerves in rats. Methods Thirty-five healthy SD rats were randomly divided into 3 groups: 5 in the control group, 15 in the simply transected optic nerve group and 15 in the optic nerve-sciatic nerve anastomosis group. The simply transected and optic nerve-sciatic nerve anastomosed models were set up, and the retinal tissues of all of the rats were taken out after 3, 7 and 14 days, respectively; and the mRNA expression of CNTF in the 3 groups were observed by semiquantitative reversal transcription-polymerase chain reaction method. Results A minimum expression of CNTF mRNA was found in the retinae of the control group, and the increased rates of expression were found in the retinae of the simple transection of optic nerve group with the increase rate of 100%, 594%, and 485% on the 3rd, 7th, and 14th day respectively after the operation, while in optic nerve-sciatic nerve anastomosis group, the increase rates were found to be 258%, 752% and 515% on the 3rd, 7th, and 14th day respectively after the operation. Conclusion Retinal neurons can respond to axonal reaction of retinal ganglion cells by up-regulate endogenous CNTF after the injury of the optic nerves, which may provide a theoretic base for the application of the exogenous CNTF. (Chin J Ocul Fundus Dis，2004，20：355-357)
Transcranial magnetic stimulation (TMS) as a noninvasive neuromodulation technique can improve the impairment of learning and memory caused by diseases, and the regulation of learning and memory depends on synaptic plasticity. TMS can affect plasticity of brain synaptic. This paper reviews the effects of TMS on synaptic plasticity from two aspects of structural and functional plasticity, and further reveals the mechanism of TMS from synaptic vesicles, neurotransmitters, synaptic associated proteins, brain derived neurotrophic factor and related pathways. Finally, it is found that TMS could affect neuronal morphology, glutamate receptor and neurotransmitter, and regulate the expression of synaptic associated proteins through the expression of brain derived neurotrophic factor, thus affecting the learning and memory function. This paper reviews the effects of TMS on learning, memory and plasticity of brain synaptic, which provides a reference for the study of the mechanism of TMS.
ObjectiveThe aim of this study was to evaluate the repair effect of spontaneous reinnervation in rats underwent recurrent laryngeal nerve (RLN) transection.
MethodsThirty male Wistar rats (340-360 g) were divided into experiment group (n=15) and blank control group (n=15), and then 15 rats of these 2 groups were divided into 3 time point groups equally:4 weeks group, 8 weeks group, and 12 weeks group. Fifteen rats of experiment group underwent right RLN transection with excision of a 5 mm segment, and other 15 rats of blank control group exposed RLN only, without transection. Grade of vocalization, maximum angle of arytenoid cartilage, axon number of distal part of RLN, and expression of the brain-derived neurotrophic factor (BDNF) in right thyroarytenoid muscle were evaluated at different time points, including 4, 8, and 12 weeks after operation.
ResultsGrade of vocalization, maximum angle of arytenoid cartilage, axon numbers of distal part of RLN, and the expression of BDNF in the right thyroarytenoid muscle of experiment group were all lower than those corresponding index of blank control group (P < 0.05), and these indexes of experiment group were restored gradually with time, but failed to reach normal level during the observed time.
ConclusionsEven though spontaneous reinnervation is presented after RLN injury, but the effect is unsatisfactory.
【Abstract】 Objective To construct tissue engineered skeletal muscle in vivo using glial cell derived neurotrophic factor (GDNF) genetically modified myoblast (Mb) on acellular collagen sponge with hypoglossal nerve implantation, and to observe whether structural or functional connection could be established between engineered tissue and motor nerve or not. Methods Mbs were isolated from 7 male Lewis rats at age of 2 days, cultured and genetically modified by recombinant adenovirus carrying GDNF cDNA (MbGDNF). Calf skin-derived acellular collagen sponge was used as scaffold; cell adhesion was detected by scanning electron microscope after 24 hours. Hypoglossal nerve was implanted into Mb-scaffold complex (Mb group, n=27) or MbGDNF-scaffold complex (MbGDNF group, n=27) in 54 female Lewis rats at age of 8 weeks. HE staining was performed at 1, 6, and 12 weeks postoperatively, and immunohistochemistry staining and fluorescence in situ hybridization were used. Results MbGDNF could highly expressed GDNF gene. Mb and MbGDNF could adhere to the scaffold and grew well. HE staining showed tight junctions between implant and peripheral tissue with new muscle fiber and no distinguished line at 12 weeks in 2 groups. Immunohistochemistry staining showed that positive cells of myogenin and slow skeletal myosin were detected, as well as positive cells of actylcholine receptor α1 at 1, 6, and 12 weeks. The positive cells of Y chromosome decreased with time. At 1, 6, and 12 weeks, the positive neurons were 261.0 ± 6.6, 227.3 ± 8.5, and 173.3 ± 9.1, respectively in MbGDNF group, and were 234.7 ± 5.5, 196.0 ± 13.5, and 166.7 ± 11.7, respectively in Mb group; significant differences were found between 2 groups at 1 and 6 weeks (P lt; 0.05), no significant difference at 12 weeks (P gt; 0.05). Conclusion Connection can be established between engineered tissue and implanted hypoglossal nerve. Recombinant GDNF produced by MbGDNF might play a critical role in protecting central motor neurons from apoptosis by means of retrograde transportation.
Objective To identify glial cell line-derived neurotrophic factor (GDNF) recombinant retroviral vector and to establish its packaging cell line PA317. Methods PA317 cells were transfected with recombinant retroviral vector pLXSN-GDNF using liposomes. The recombinant retroviral particles were then harvested from culture media of G418 resistant transfected cells and analyzed using RT-PCR. Virus titers in supernatants were investigated. Results Sequencing date indicated that GDNF gene was exactly identical to the sequence in the GeneBank. PA317 cells were transfected with recombinant retroviral vector pLXSN-GDNF using liposomes, and virus titers insupernatants harvested from culture media of G418 resistant transfected cells were 104-105 CFU/ml. Conclusion Packaging cell line PA317/pLXSN-GDNF was established.
